Connexin43 (Cx43) is the most abundant gap junction protein in the heart and plays a critical role in heart development. Cx43-/- mice develop right ventricular outflow tract (RVOT) obstruction in utero and die of pulmonary oligemia shortly after birth. The molecular mechanisms, the critical period in heart development and the specific cell lineages responsible for the defect in heart formation are unknown. We hypothesize that the Cx43-/- phenotype results from a primary developmental defect in cells of neural crest origin, rather than in the myocytes themselves. Furthermore, we hypothesize that the loss of Cx43 results in dysregulated cell proliferation, a phenomenon that may underlie the disordered neural crest-derived tissue growth causing RVOT obstruction in Cx43-/- mice. To test these hypotheses, we will conditionally inactivate the Cx43 gene in a lineage-specific manner. We predict, according to our hypothesis, that targeted inactivation of the Cx43 gene in cells of neural crest origin will recapitulate the phenotype of the Cx43-/- mouse. For this proposal, we have engineered murine embryonic stem (ES) cells to harbor loxP sites flanking the Cx43 open reading frame (floxed) for Cre recombinase-mediated inactivation of the Cx43 gene. We have established that our lines of targeted ES cells express wildtype levels of Cx43 mRNA and protein. In addition, upon transfection with a vector containing the Cre cDNA or infection with a Cre-expressing adenovirus, the floxed Cx43 gene recombines and is inactivated as predicted. As a result, we have generated chimeric mice carrying the mutated Cx43 allele with one of our targeted ES cell lines. Using these reagents, we propose to study, through inducible loss-of-function experiments, whether a specific lineage is responsible for the Cx43-/- developmental phenotype. Growth and differentiation characteristics of Cx43-/- ES cells and proliferation of Cx43-/- cells in chimeric mice will also be studied. In order to determine when Cx43 is required during embryogenesis, we are developing a novel, reversible, tetracycline (TCN)-regulated "knock-in" system to control endogenous Cx43 gene expression. The proposed experiments promise to provide novel information delineating the molecular mechanisms by which loss of Cx43 results in cardiac dysmorphogenesis. These results will add important information to our knowledge of developmental biology, while also elucidating potential mechanisms of congenital heart disease.